Methods of purifying a nucleic acid and formulation and kit for use in performing such methods

ABSTRACT

A formulation containing guanidine thiocyanate or guanidine hydrochloride together with acetamide, one or more acetamide derivatives, or a combination of acetamide and one or more acetamide derivatives is provided with methods to use the formulation to purify one or more nucleic acids contained in a medium.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 12/549,806filed Aug. 28, 2009, now issued as U.S. Pat. No. 8,039,613 the contentof which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to methods of purifying one or more nucleicacids, and also to a formulation and kit for use in performing suchmethods.

BACKGROUND OF THE INVENTION

The purification of nucleic acids plays an important role in scientificprocedures. There are a number of known methods of purifying single- anddouble-stranded DNA contained in biological fluids such as human blood,serum, and cultured cells, as well as plants, animal and human tissues,and other specimens. However, such methods can result in very low yieldsand do not always work well when trying to extract small amounts ofnucleic acids from large samples. Known methods are described in, forexample, Nargessi, U.S. Pat. No. 6,855,499 (2005); Tereba et al., U.S.Pat. No. 6,673,631 (2004); McKernan et al., U.S. Pat. No. 6,534,262(2003); Taylor et al., J. Chromatograpy A, 890:159-166 (2000); Ahn etal., BioTechniques, 29:466-468 (2000); Scott Jr. et al., Lett. Appi.Microbiol., 31:95-99 (2000); Lin et al., BioTechniques, 29:460-466(2000); Smith et al., U.S. Pat. No. 6,027,945 (2000); Mrazek et al.,Acta Univ. Palacki. Olomuc., Fac. Med. 142:23-28 (1999); Hawkins, U.S.Pat. No. 5,898,071 (1999); and Hawkins, U.S. Pat. No. 5,705,628 (1998).

SUMMARY OF THE INVENTION

The present invention represents an improvement over the known methodsdescribed in the aforementioned literature.

In one aspect, the invention relates to a method of purifying at leastone nucleic acid, such as deoxyribonucleic acid (DNA), ribonucleic acid(RNA), and/or peptide nucleic acid (PNA), which is contained in amedium, such as whole blood, plasma, or tissue cell cultures obtainedfrom humans, plants, or animals. The method includes steps of (a)combining the medium containing the at least one nucleic acid with atleast one binding matrix and a formulation in order to cause the atleast one nucleic acid to separate from its in vivo cellular environmentand bind to at least one binding matrix, (b) separating the bindingmatrix with at least one nucleic acid bound thereto from substantiallythe rest of the combined medium and formulation, and (c) eluting the atleast one nucleic acid from the binding matrix, thereby obtaining the atleast one nucleic acid in a substantially purified form.

A nucleic acid is considered to be in a “substantially purified form”when the nucleic acid has been separated from its in vivo cellularenvironment and obtained in a form that is useful in one or morescientific procedures, such as the isolation of genetic material,polymerase chain reactions, electrophoresis, sequencing, and cloning,among others.

The formulation used in the foregoing method contains an amount ofguanidine thiocyanate and an amount of (i) acetamide, (ii) one or moreacetamide derivatives, or (iii) a combination of acetamide and one ormore acetamide derivatives. Preferred acetamide derivatives includemethylacetamide and dimethylacetamide. Herein, guanidine thiocyanate issometimes referred to as “GTC,” and the combination of guanidinethiocyanate with acetamide and/or one or more acetamide derivatives issometimes referred to as “GTC-A.”

In the above method, the respective amounts of GTC and acetamide and/oracetamide derivative(s) present in the formulation are sufficient tocause the at least one nucleic acid to separate from its in vivocellular environment and bind to the binding matrix. Preferably, theconcentration of GTC in the formulation is from approximately 1.7M toapproximately 4.3M, more preferably from approximately 4.0M toapproximately 4.3M. Preferably, the concentration of acetamide and/oracetamide derivative(s) in the formulation is from approximately 5.0M toapproximately 7.5M, more preferably from approximately 5.0M toapproximately 7.1M.

Any of a number of known binding matrices can be used in the foregoingmethod, depending on the type of nucleic acids sought to be purified.Those skilled in the art will be able to select binding matrices thatare compatible with the nucleic acid of interest. Examples of suitablebinding matrices include, but are not limited to, paramagnetic celluloseparticles, paramagnetic carboxy-cellulose particles, paramagnetic citruspectin particles, paramagnetic apple pectin particles, paramagneticzeolite particles, paramagnetic silica particles, cellulose membranes,silica membranes, cellulose acetate columns, nylon membrane columns,PVDF membrane columns, polypropylene columns, HIGH PURE™ spin columns(available from Roche-Diagnostics, item 1 828 665), and clearingcolumns.

The ratio of formulation to medium used in the above method ispreferably from 1:1 to 30:1, more preferably from 1.5:1 to 8:1, byvolume. The ratio of binding matrix to medium is preferably from 0.005:1to 0.5:1, more preferably from 0.2:1 to 0.4:1, by volume. One skilled inthe art will be able to select proportions, within or outside of thesepreferred ranges, depending on the nucleic acid(s) of interest, theconcentration of the formulation, and the type of binding matrixemployed, among other variables. Therefore, the invention is not limitedto these preferred ranges.

Optionally, one or more additional ingredients can be combined with themedium, the binding matrix, and the formulation. For example, one ormore enzymes which aid in the degradation and lysis of cellularstructure can be used to facilitate the separation of nucleic acids fromtheir mediums. Examples of suitable additional ingredients include, butare not limited to, proteinase K (available from Promega, catalog itemV3021), beta-mercaptoethanol (BME), tris(carboxyethyl)phosphine (TCEP),dithiothreitol (DTT) (available from Sigma, catalog item 43815),1-thioglycerol (1-TG) (available from SIGMA-ALDRICH™, catalog itemM2172), digitonin, lysis solutions, CHAPS(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; availablefrom SIGMA-ALDRICH™, catalog item C3023), TERGITOL™ type NP-9(26-(4-nonylphenoxy)-3,6,9,12,15,18,21,24-octaoxahexacosan-1-ol;available from SIGMA-ALDRICH™, catalog item np9), and TRITON™ X-100(4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol; available fromThermo Scientific, Waltham, Mass., catalog item BP151). How theseadditional ingredients are employed is not critical. For example, theycan be incorporated in the formulation or they can be added to themedium either before or after the medium is combined with the bindingmatrix and/or the formulation.

This method can also be used to purify at least two different nucleicacids contained in a single medium. This involves combining a mediumcontaining at least two different nucleic acids with a first bindingmatrix compatible with a first nucleic acid contained in the medium, asecond binding matrix compatible with a second nucleic acid contained inthe medium, and a GTC-A formulation as described above. This causes thefirst and second nucleic acids to separate from their in vivo cellularenvironments and to bind to the first and second binding matrices,respectively. Each of the first binding matrix with the first nucleicacid bound thereto and the second binding matrix with the second nucleicacid bound thereto then is separated from substantially the rest of thecombined medium and formulation. Further, the first nucleic acid iseluted from the first binding matrix, and the second nucleic acid iseluted from the second binding matrix, thereby obtaining each of thefirst and second nucleic acids in a substantially purified form.

Optionally, a medium containing the second nucleic acid and theformulation can be transferred after the first nucleic acid contained inthe medium binds to the first binding matrix. This is followed by atransfer of the remaining medium to the second binding matrix compatiblewith the binding of the second nucleic acid contained in the medium,before separating each of the first and second nucleic acids from thefirst and second binding matrices, respectively.

In another aspect, the invention relates to a method as described above,except that the final eluting step is not necessarily required, althoughsuch a step is not excluded. Thus, the binding matrix with the nucleicacid bound thereto can be stored and/or transported for later use inscientific procedures, which may or may not involve eluting the nucleicacid from the binding matrix.

In another aspect, the invention relates to a kit for use in purifyingnucleic acids and/or binding nucleic acids to a binding matrix. The kitincludes a binding matrix and a GTC-A formulation as described above.Preferably, the ratio of binding matrix to formulation in the kit iswithin the range of from 1:400 to 1:1, by volume. The ratio of bindingmatrix to formulation can be varied within or outside of this preferredrange depending on, among other things, the nucleic acid(s) of interestand the type of binding matrix employed.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a photograph showing the results of an electrophoresisanalysis performed in accordance with Example 2, described below.

FIG. 2 is a photograph showing the results of an electrophoresisanalysis performed in accordance with Examples 3 and 4, described below.

FIGS. 3A and 3B are photographs showing the results of anelectrophoresis analysis performed in accordance with Example 5,described below.

FIG. 4 is a photograph showing the results of an electrophoresisanalysis performed in accordance with Example 6, described below.

FIG. 5 is a photograph showing the results of an electrophoresisanalysis performed in accordance with Example 7, described below.

FIG. 6 is a photograph showing the results of an electrophoresisanalysis performed in accordance with Example 8, described below.

FIGS. 7A and 7B are photographs showing the results of anelectrophoresis analysis performed in accordance with Example 9,described below.

FIG. 8 is a photograph showing the results of an electrophoresisanalysis performed in accordance with Example 10, described below.

FIGS. 9A and 9B are photographs showing the results of anelectrophoresis analysis performed in accordance with Example 11,described below.

FIG. 10 is a photograph showing the results of a blood card prepared inaccordance with Example 12, described below.

FIG. 11 is a photograph showing the results of an electrophoresisanalysis performed in accordance with Example 12, described below.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following are non-limiting examples of preferred implementations ofthe present invention. Throughout, all volumes, pH levels, andconcentrations are at room temperature unless stated otherwise.

Nucleic Acids

The nucleic acids capable of being purified using the present inventioninclude, but are not limited to DNA (single-stranded, double-stranded,covalently closed, and relaxed circular forms), RNA (single-stranded anddouble-stranded), PNA, and hybrids of the foregoing.

Nucleic-Acid-Containing Mediums

As used herein, the term “medium” encompasses any biological material,either naturally occurring or scientifically engineered, that containsat least one nucleic acid in addition to other non-nucleic acidmaterial, such as biomolecules (e.g., proteins, polysaccharides, lipids,low molecular weight enzyme inhibitors, oligonucleotides, primers,templates), polyacrylamide, trace metals, organic solvents, etc.Examples of naturally-occurring mediums include, but are not limited to,whole blood, plasma, and other body fluids, as well as tissue cellcultures obtained from humans, plants, or animals. Examples ofscientifically-engineered mediums include, but are not limited to,lysates, nucleic acid samples eluted from agarose and/or polyacrylamidegels, solutions containing multiple species of DNA molecules resultingeither from a polymerase chain reaction (PCR) amplification or from DNAsize selection procedures, and solutions resulting from post-sequencingreactions.

Binding Matrix

Advantageously, one or more binding matrices can be used in the presentinvention. As used, herein, the term “binding matrix” encompasses anyform capable of binding a nucleic acid. Those skilled in the art will beable to select an appropriate binding matrix for the nucleic acid(s) ofinterest.

Examples of suitable binding matrices include MAGAZORB™ paramagneticparticles (available from Promega, Madison, Wis., catalog item MB1001),GENFIND™ particles (available from BECKMAN-COULTER™, Fullerton, Calif.),MAGNESIL™ Blue paramagnetic silica particles (available from Promega,catalog item A2201), zeolite particles (see Bitner et al., U.S.Published Patent Appln. No. 2007/0172855, the entirety of which isincorporated by reference herein), and paramagnetic apple or citruspectin particles (see Example 1 below). Other suitable binding matricesinclude, without limitation, paramagnetic silica particles, cellulosemembranes, silica membranes, or columns such as cellulose acetatecolumns, nylon membrane columns, PVDF membrane columns, polypropylenecolumns, pure spin columns, and clearing columns for use in DNA IQ™ spinbaskets (available from Promega, catalog item V1221). More specifically,suitable columns include DNA-IQ™ columns, SV columns, Corning spincolumns (available from Corning of Corning, N.Y., catalog item 8160),nylon membrane Corning spin columns (available from Corning, catalogitem 8169), PVDF membrane in a polypropylene spin columns (availablefrom Millipore Ultrafree-MC of Beverly, Mass., catalog item VFC3OGVNB),polypropylene membrane (available from Corning, catalog item AN0604700)in a DNA-IQ™ column, HIGH PURE™ Spin Filter tubes (available from Rocheof Indianapolis, Ind., catalog item 1828665), and clearing columns(available from Promega, catalog item Z568A). Schleicher & Schuellcellulose cards (available from Keene of NH, catalog item GB003) alsoprovide a useful binding matrix, particularly when storing and/ortransporting nucleic acids for later use in downstream procedures.

The ratio of binding matrix to medium is preferably from 0.005:1 to0.5:1, more preferably from 0.2:1 to 0.4:1, by volume. One skilled inthe art will be able to select optimal proportions, within or outside ofthese preferred ranges, depending on the nucleic acid(s) of interest andthe type of binding matrix, among other factors.

GTC-A Formulation

The GTC-A formulation acts as a lysis and/or binding solution thatseparates the nucleic acid of interest from its in vivo cellularenvironment and, if a binding matrix is present, facilitates the bindingof the nucleic acid to the binding matrix. As mentioned above, theformulation contains an amount of GTC and an amount of acetamide and/orone or more acetamide derivatives. The GTC and acetamide and/or one ormore acetamide derivatives may be added together, or sequentially to thesample medium. As an example of sequential addition, the GTC may beadded to the sample medium in a first solution, for example, to lysecells, and the acetamide and/or one or more acetamide derivatives may beadded in a second solution, for example, as a binding solution topromote binding of the DNA or RNA to the binding matrix.

As demonstrated by the examples below, other salts or salt and amidecombinations, such as guanidine hydrochloride (available from Promega,catalog item H5381), guanidine hydrochloride and acetamide, GTC alone,or acetamide alone, are relatively ineffective in the purification ofnucleic acids. Surprisingly, even though neither GTC alone nor acetamidealone is effective in purifying nucleic acids, the combination of GTCand acetamide and/or acetamide derivative(s) is highly effective.

The respective amounts of GTC and acetamide and/or acetamidederivative(s) present in the GTC-A formulation can be adjusted tovarious concentrations. The concentration of GTC in the formulation ispreferably from approximately 1.7M to approximately 4.3M, morepreferably from approximately 4.0M to approximately 4.3M. Theconcentration of acetamide and/or acetamide derivative(s) in theformulation is preferably from approximately 5.0M to approximately 7.5M,more preferably from approximately 5.0M to approximately 7.1M.

The proportion of formulation to medium depends on a number ofvariables, including, without limitation, the concentration of theformulation, the nucleic acid(s) of interest, whether a binding matrixis used, and, if so, what type. A preferred range of ratios offormulation to medium is from 1:1 to 30:1, by volume. A more preferredrange is from 1.5:1 to 8:1, by volume.

GTC can be purchased from Promega, catalog item V2791. Acetamide can bepurchased from SIGMA-ALDRICH™, catalog item A0500-500G. As mentioned,GTC can also be used with derivatives of acetamide. Preferred acetamidederivatives include N-methylacetamide (available from ACROS of FairLawn, N.J., catalog item 126141000) and N,N-dimethylacetamide (availablefrom SIGMA-ALDRICH™, catalog item D5511). Sometimes, such as whenpurifying RNA from HEK293 tissue cells, the use of GTC andN,N-dimethylacetamide is preferred over the use of GTC and acetamide orGTC and N-methylacetamide.

Optionally, the GTC-A formulation can further contain one or moreadditional ingredients such as, for example, proteinase K,beta-mercaptoethanol, tris(carboxyethyl)phosphine, dithiothreitol, 1-TG,digitonin, lysis solutions, CHAPS, TERGITOL™ type NP-9, and TRITON™X-100.

In addition to being used as a lysis and/or binding solution, the GTC-Aformulation can also be used as a wash solution for removing impurities,as described in the methods below.

Kits

The GTC-A formulation can be combined with one or more binding matricesin a kit that can be used in the purification of nucleic acids.Preferably, the ratio of binding matrix to formulation in the kit iswithin the range of from 1:400 to 1:1, by volume. The ratio of bindingmatrix to formulation can be varied within or outside of this preferredrange, depending on the specific contents of the kit and the applicationfor which the kit is intended. The kit may comprise GTC in a firstcontainer, and acetamide and/or one or more acetamide derivatives in asecond container, combined with one or more binding matrices in a kitthat can be used in the purification of nucleic acids. Alternatively,the GTC and acetamide and/or one or more acetamide derivatives may becombined in a single container. The one or more binding matrices may becombined within one of the above containers, or provided as a separateitem in a kit that can be used in the purification of nucleic acids.

Advantageously, the kit can include more than one type of bindingmatrix, each compatible with a different type of nucleic acid. In thatcase, the kit can be used in the selective purification of differenttypes of nucleic acids.

First Method

In one preferred implementation, the invention relates to a method ofpurifying at least one nucleic acid contained in a medium. The mediumcontaining the at least one nucleic acid is combined with at least onebinding matrix and a GTC-A formulation in order to cause the at leastone nucleic acid to separate from its in vivo cellular environment andbind to at least one binding matrix. The medium, binding matrices, andGTC-A formulation can be combined in any order or simultaneously. Thebinding matrices with at least one nucleic acid bound thereto then isseparated from substantially the rest of the combined medium andformulation, for example, by using a magnetic rack, by centrifuging, orby filtration. Optionally, at this point, bound binding matrix and thenucleic acid combinations can be washed using any suitable washsolution, including the GTC-A formulation, in order to remove anyimpurities. Thereafter, the at least one nucleic acid is eluted from thebinding matrix, thereby obtaining the at least one nucleic acid in asubstantially purified form. This elution step can immediately followthe aforementioned steps, or it can be performed at a later time. Bychoosing to elute the nucleic acid from the binding matrix at a latertime, the nucleic acids can be stored for downstream activities.

In more detail, the elution step uses an elution buffer to separate thenucleic acid from the binding matrix, after which the substantiallypurified nucleic acid is contained in the elution buffer. Suitableelution buffers include, but are not limited to, nuclease-free water oraqueous solutions such as, for example, TRIS™-HCl, Tris-acetate,sucrose, and formamide solutions. A preferred elution buffer is a TRIS™buffer with ethylenediaminetetraacetic acid (EDTA). More preferably, theelution buffer is about 10 mM TRIS™ (pH 8.0) and about 1 mM EDTA HEPES™(pH 7.5). Elution of the nucleic acid from the binding matrix occursquickly (e.g., in thirty seconds or less) when a suitable low ionicstrength elution buffer is used.

Following purification, the nucleic acids can be used in any of a numberof known scientific procedures, including, without limitation, theisolation of genetic material, polymerase chain reactions,electrophoresis, sequencing, cloning, and the like.

This method can also be used to purify at least two different nucleicacids contained in a single medium. This involves combining a mediumcontaining at least two different nucleic acids with a first bindingmatrix compatible with binding a first nucleic acid contained in themedium, and a second binding matrix compatible with binding a secondnucleic acid contained in the medium, and a GTC-A formulation asdescribed above. This causes the first and second nucleic acids toseparate from their in vivo cellular environments and to bind to thefirst and second binding matrices, respectively. Each of the firstbinding matrix with the first nucleic acid bound thereto and the secondbinding matrix with the second nucleic acid bound thereto then isseparated from substantially the rest of the combined medium andformulation. Then, the first nucleic acid is eluted from the firstbinding matrix, and the second nucleic acid is eluted from the secondbinding matrix, thereby obtaining each of the first and second nucleicacids in a substantially purified form. Alternatively, this methodinvolves combining a medium containing at least two different nucleicacids with a first binding matrix compatible with binding a firstnucleic acid contained in the medium (e.g DNA-IQ™ particles which arecompatible with binding DNA), followed by a transfer of the remainingsolution depleted of the first nucleic acid (e.g., the RNA remainingafter DNA binding to DNA-IQ™ particles) to a second binding matrix(e.g., paramagnetic zeolite particles) compatible with binding of theremaining nucleic acids, thereby separately obtaining each of the firstand second nucleic acids in a substantially purified form.

Second Method

In another implementation, the invention relates to a method asdescribed above, except that this method does not necessarily requireeluting the nucleic acid from the binding matrix. Rather, the bindingmatrix with the nucleic acid(s) bound thereto can be stored and/ortransported for later use in scientific procedures, which may or may notinvolve eluting the nucleic acid(s) from the binding matrix.

This method is particularly useful when using the Schleicher & Schuellcellulose card as the binding matrix. When there is a need for purifiednucleic acids, a hole can be punched from the cellulose card and thenucleic acid bound thereto can be eluted for use in a scientificprocedure.

Example 1

In this example, paramagnetic apple or citrus pectin particles suitablefor use in certain embodiments of the present invention as a bindingmatrix were prepared as follows:

-   -   1. In a beaker, mix 0.5 gm of Fe₃O₄ particles (magnetite,        available from SIGMA-ALDRICH™ of St. Louis, Mo., catalog item        31,006-9) with 1.0 gm of apple pectin particles (available from        SIGMA-ALDRICH™, catalog item P8471) or citrus pectin particles        (available from SIGMA-ALDRICH™, catalog item P9135) in 8 ml of        water.    -   2. Adjust the pH of the mixture, first by adding 3.0 ml of 56%        KOH. After mixing for 5 minutes at about 21° C., lower the pH by        adding 3.3 ml of 3.0M HCl, with periodic testing of the        mixture's pH using pH paper. The resulting pH should be about 3.        Next, add 3.5 ml of 1.32M KOAc having a pH of 4.8 so that the        resulting pH of the mixture is between 4 and 5.    -   3. Allow the mixture to sit overnight at about 21° C. Some large        (e.g., 1-2 cm³) particles will form and settle to the bottom of        the beaker. Pour off the rest of the mixture into a tube, and        label the beaker containing the large particles as “large.”    -   4. Place the tube containing the rest of the mixture on a        magnetic rack and magnetize the mixture. After 2 minutes, pour        off the portion of the mixture that has not been attracted to        the side wall of the tube into another tube. Label the tube        containing the particles that were attracted to the side wall as        “main.” There should be approximately 3 ml of “main” particles,        ranging anywhere from about 0.6 μm to about 80 μm in size.    -   5. Place the tube containing the rest of the mixture back on the        magnetic rack and magnetize the mixture. After 10 minutes, pour        off and discard the portion of the mixture that has not been        attracted to the side wall of the tube. Label the tube        containing the particles that were attracted to the side wall as        “first cut.” There should be approximately 1.5 ml of “first cut”        particles.    -   6. Wash the “large,” “main,” and “first cut” particles three        times with nanopure water to remove any residual KOAc and/or        residual pectin particles. For each wash, add 10 ml of the        nanopure water to the set of particles, allow the particles to        settle, and then pour off the nanopure water.

Any of the three sets of paramagnetic pectin particles—“large,” “main,”and “first cut”—can be used as a binding matrix; however, in theexamples that follow, only the “main” particles are used.

Example 2

In this example, nine different lysis-binding formulations were used,some more successfully than others, in an attempt to purify DNA fromhuman whole blood samples. The following procedure was used:

-   -   1 Prepare nine samples, 1-9, by adding 50 μl of MAGAZORB™        paramagnetic particles, followed by 200 μl of human whole blood        (available from Bioreclamation, Inc., Hicksville, N.Y., catalog        item HMPLEDTA3), to each of nine 1.5 ml plastic tubes.    -   2. Add 800 μl of one of the following nine formulations to a        different one of samples 1-9 as follows: (a) to sample 1, add        5.0M acetamide; (b) to sample 2, add 2.6M guanidine        hydrochloride (GHCl); (c) to sample 3, add a mixture of 2.6M        GHCl and 5.0M acetamide; (d) to sample 4, add 2.6M GTC; (e) to        sample 5, add a mixture of 2.6M GTC and 5.0M acetamide; (f) to        sample 6, add a mixture of 4.3M GHCl and 5.0M acetamide; (g) to        sample 7, add 6.5M GHCl; (h) to sample 8, add 9.0M acetamide;        and (i) to sample 9, add a mixture of 4.3M GTC and 5.0M        acetamide. Mix each sample thoroughly by repeated pipetting.    -   3. Allow each sample to sit for 10 minutes at about 21° C., and        then place each sample on a magnetic rack in order to separate        the MAGAZORB™ paramagnetic particles from the rest of the        sample. Remove and discard the supernatants, leaving only the        MAGAZORB™ paramagnetic particles.    -   4. Remove each of samples 1-9 from the magnetic rack and wash        the MAGAZORB™ paramagnetic particles with 800 μl of RNA Wash        Solution (available from Promega, catalog item Z3091), mixing        the particles into the solution by pipetting. Place each sample        back on the magnetic rack in order to separate the MAGAZORB™        paramagnetic particles from the rest of the sample. Again,        remove and discard the supernatants, leaving only the MAGAZORB™        paramagnetic particles.    -   5. Repeat step 4 in order to wash the MAGAZORB™ paramagnetic        particles a second time.    -   6. Allow samples 1-9 to air dry on the magnetic rack for 20        minutes at a temperature of about 21° C.    -   7. Remove the samples from the magnetic rack and elute the DNA        from the MAGAZORB™ paramagnetic particles by adding 200 μl of        nuclease-free water at a temperature of about 56° C. to each        sample. Allow the samples to elute for 20 minutes, occasionally        vortexing each sample to release the DNA from the MAGAZORB™        paramagnetic particles into the nuclease-free water, and then        place the samples back on the magnetic rack.    -   8. Using blue/orange 6× loading dye (available from Promega,        catalog item G190), load 10 μl of the DNA-containing,        nuclease-free water from each of samples 1-9 into a respective        one of nine agarose gel electrophoresis lanes containing a 15%        TBE-urea gel (available from INVITROGEN™, Carlsbad, Calif.,        catalog item EC68852BOX). To a tenth lane, add nothing. To an        eleventh lane, add 10 μl of genomic DNA standard (available from        Promega, catalog item G3041) to serve as a control. To a twelfth        lane, add 10 μl of 100 bp DNA ladder of standard molecular        weight (available from Promega, catalog item G2101) to serve as        a scale.    -   9. Perform electrophoresis using bromphenol blue dye at 120        volts for about 3 hours or until the bromphenol blue dye reaches        the bottom slit of the gel. Stain each lane for 15 minutes with        SYBR™ Gold (available from INVITROGEN™, catalog item S11494),        and digitally image all of the lanes using an ALPHA INNOTECH        FLUOROCHEM™ Imaging System, and, in particular, the Amersham        TYPHOON™ platform with settings of: 1. ex488/em526. 2. PMT 450.

FIG. 1 shows the results of the electrophoresis analysis of the samplesprepared according to Example 2. In FIG. 1, the lanes are numbered 1-12,from left to right. Lanes 1-9 show the results for samples 1-9,respectively; lane 10 contains no sample; lane 11 shows the genomic DNAstandard; and lane 12 shows the 100 bp DNA ladder of standard molecularweight.

As demonstrated in FIG. 1, in the case of human whole blood, the use ofacetamide alone (lanes 1 and 8), GHCl alone (lanes 2 and 7), GTC alone(lane 4), or the combination of acetamide and GHCl (lanes 3 and 6) didnot result in substantial DNA purification. Surprisingly, even thoughneither GTC alone nor acetamide alone was effective, using a combinationof GTC and acetamide (lanes 5 and 9) resulted in substantial DNApurification.

Example 3

In this example, DNA from human whole blood samples was purified using aGTC-A formulation together with each of four different binding matricesusing the following procedure:

-   -   1. Prepare eight samples, 1-8, by adding 800 μl of “L₁,” which        consists of 2.7M GTC and 6.8M acetamide, to each of eight 1.5 ml        plastic tubes.    -   2. To each sample, add 20 μl of 20 mg/ml proteinase K and mix        the sample by pipetting.    -   3. To each sample, add 100 μl of human whole blood and mix six        times by pipetting.    -   4. Incubate each sample for five minutes at a temperature of        about 56° C., mix each sample by pipetting, and then incubate        each sample for an additional five minutes at about 56° C.    -   5. Add one of the four aforementioned binding matrices to each        of samples 1-8 as follows: (a) to each of samples 1 and 2, add        40 μl (1.9 mg) of MAGAZORB™ paramagnetic particles; (b) to each        of samples 3 and 4, add 3 μl of paramagnetic carboxy-cellulose        GENFIND™ particles; (c) to each of samples 5 and 6, add 40 μl        (2.8 mg) of “main” paramagnetic citrus pectin particles (from        Example 1); and (d) to each of samples 7 and 8, add 40 μl (4.2        mg) of MAGNESIL™ Blue paramagnetic silica particles. Mix each        sample by pipetting and let the samples stand for 10 minutes at        a temperature of about 21° C.    -   6. Magnetize the samples by placing them on a magnetic rack.        Remove the excess fluid, so that only the magnetized particles        remain for each sample.    -   7. Remove the samples from the magnetic rack and wash each        sample by adding 500 μl of L₁ and mixing by pipetting. Next,        magnetize the samples by placing them back on the magnetic rack.        Remove the excess fluid, so that only the magnetized particles        remain for each sample.    -   8. Repeat the wash a second and third time using 500 μl of L₁ by        following the procedure of step 7.    -   9. Wash each sample one additional time using 500 μl of Alcohol        Wash, Blood (available from Promega, catalog item MD1411) by        following the above wash procedure of step 7, except        substituting the Alcohol Wash, Blood for L₁.    -   10. Allow the samples to air dry on the magnetic rack for 10        minutes at a temperature of about 21° C.    -   11. Remove the samples from the magnetic rack and elute the DNA        from the particles by adding 200 μl of 10 mM TRIS™ HCl (pH 8.0)        to each sample. Allow the samples to elute for 10 minutes,        occasionally vortexing each sample to release the DNA from the        particles into the TRIS™ HCl, and then place the samples on a        magnetic rack.    -   12. Using blue/orange 6× loading dye, load 8 μl of the        DNA-containing TRIS™ HCl from each of samples 1-8 into a        respective one of eight agarose gel electrophoresis lanes        containing a 15% TBE-urea gel.    -   13. Perform electrophoresis using bromphenol blue dye at 120        volts for about 3 hours or until the bromphenol blue dye reaches        the bottom slit of the gel. Stain each lane for 15 minutes with        SYBR™ Gold, and digitally image all of the lanes using an ALPHA        INNOTECH FLUOROCHEM™ Imaging System, and, in particular, the        Amersham TYPHOON™ platform with settings of: 1. ex488/em526. 2.        PMT 450.

FIG. 2 shows the results of the electrophoresis analysis of the samplesprepared according to Example 3. In FIG. 2, the lanes are numbered 1-12,from left to right. Lanes 1-8 show the results for samples 1-8,respectively. (Lanes 9-12 relate to Example 4, which is discussedbelow.)

As shown in FIG. 2, in the case of human whole blood, all of the samplesprepared in accordance with Example 3 achieved substantial purificationof genomic DNA. The smaller amount of DNA obtained with the paramagneticcarboxy-cellulose GENFIND™ particles (lanes 3 and 4) is believed to beattributable to the small amount (3 μl) of particles used per sample.

Example 4

In this example, DNA from human whole blood samples was released using aGTC-A formulation together with one of two different binding matrices.The following procedure was used:

-   -   1. Prepare four samples, 1-4, by adding 800 μl of “D_(X),” which        consists of 4.0M GTC, 5.0M acetamide, 20% (volume/volume) 1-TG,        0.64% (weight/volume) CHAPS, 0.64% (volume/volume) TERGITOL™        type NP-9, and 0.16% (volume/volume) TRITON™ X-100, to each of        four 1.5 ml plastic tubes.    -   2. To each sample, add 20 μl of 20 mg/ml proteinase K and mix        the sample by pipetting.    -   3. To each sample, add 500 μl of human whole blood and mix ten        times by pipetting.    -   4. Incubate each sample for 10 minutes at about 21° C., mix each        sample by pipetting, and then incubate each sample for an        additional 10 minutes at about 21° C.    -   5. Add one of the following binding matrices to samples 1-4 as        follows: (a) to each of samples 1 and 2, add 100 μl (4.8 mg) of        MAGAZORB™ paramagnetic particles; and (b) to each of samples 3        and 4, add 100 μl (7 mg) of “main” paramagnetic citrus pectin        particles (from Example 1). Mix each sample by pipetting and        allow the samples to sit for 10 minutes at about 21° C.    -   6. Magnetize the samples by placing them on a magnetic rack.        Remove the excess fluid, so that only the magnetized particles        remain for each sample.    -   7. Remove the samples from the magnetic rack and wash each        sample by adding 500 μl of D_(X) and mixing by pipetting. Next,        magnetize the samples by placing them back on the magnetic rack.        Remove the excess fluid, so that only the magnetized particles        remain for each sample.    -   8. For each sample, repeat the wash procedure of step 7 eight        more times using 500 μl of D_(X) each time.    -   9. Wash each sample twice with 500 μl of Alcohol Wash, Blood        using the wash procedure of step 7, except substituting the        Alcohol Wash, Blood for D_(X).    -   10. Allow the samples to air dry on the magnetic rack for 20        minutes at about 21° C.    -   11. Remove the samples from the magnetic rack and elute the DNA        from the particles by adding 200 μl of 10 mM TRIS™ HCl (pH 8.0)        to each sample. Allow the samples to elute for 10 minutes,        occasionally vortexing the samples to release the DNA from the        particles into the TRIS™ HCl, and place the samples on a        magnetic rack.    -   12. Using blue/orange 6× loading dye, load 8 μl of the        DNA-containing TRIS™ HCl from each of samples 1-4 into a        respective one of four agarose gel electrophoresis lanes        containing a 15% TBE-urea gel.    -   13. Perform electrophoresis using bromphenol blue dye at 120        volts for about 3 hours or until the bromphenol blue dye reaches        the bottom slit of the gel. Stain each lane for 15 minutes with        SYBR™ Gold, and digitally image all of the lanes using an ALPHA        INNOTECH FLUOROCHEM™ Imaging System, and, in particular, the        Amersham TYPHOON™ platform with settings of: 1. ex488/em526. 2.        PMT 450.

Lanes 9-12 of FIG. 2 show the results of the electrophoresis analysis ofsamples 1-4, respectively. (Lanes 1-8 of FIG. 2 relate to Example 3,discussed above.)

As shown in FIG. 2, in the case of human whole blood, all of the samplesprepared in accordance with Example 4 achieved substantial DNApurification. Purification using the MAGAZORB™ paramagnetic particlesaveraged 4.5 μg of DNA (lanes 9 and 10) and purification using the“main” paramagnetic citrus pectin particles averaged 4.9 μg of DNA(lanes 11 and 12).

Example 5

In this example, RNA from human blood plasma samples was purified usingdifferent GTC-A formulations containing selected inhibitors of RNaseactivity. The following procedure was used:

-   -   1. In this example, twenty-three samples, 1-23, are prepared in        twenty-three 1.5 ml plastic tubes.    -   2. Prepare samples 3-12 and 16-22 by adding, to each of        seventeen of the tubes, 800 μl of a formulation consisting of        4.3M GTC, 6.0M acetamide, 0.8% CHAPS (weight/volume), 0.8%        TERGITOL™ np-9 (volume/volume), 0.2% TRITON™ X-100        (volume/volume), and the indicated one of the following        inhibitors to the final concentration indicated: (a) for sample        3, 0.008% BME (volume/volume); (b) for sample 4, 0.016% BME        (volume/volume); (c) for sample 5, 0.032% BME        (volume/volume); (d) for sample 6, 0.064% BME        (volume/volume); (e) for sample 7, 12.5 mg/ml TCEP; (f) for        sample 8, 25 mg/ml TCEP; (g) for sample 9, 50 mg/ml TCEP; (h)        for sample 10, 100 mg/ml TCEP; (i) for sample 11, 125 mM        DTT; (j) for sample 12, 200 mM DTT; (k) for sample 16, 12.5%        1-TG (volume/volume); (1) for sample 17, 20% 1-TG        (volume/volume); (m) for sample 18, 33% 1-TG        (volume/volume); (n) for sample 19, 330 mM DTT; (o) for sample        20, a mixture of 12.5% 1-TG (volume/volume) and 0.016% BME        (volume/volume); (p) for sample 21, a mixture of 20% 1-TG        (volume/volume) and 0.032% BME (volume/volume); and (q) for        sample 22, a mixture of 12.5% 1-TG (volume/volume) and 125 mM        DTT.    -   3. To each of samples 3-12 and 16-22, add 1.0 μl of luciferase        control RNA (available from Promega, catalog item L4561) and mix        the sample by pipetting.    -   4. To each of samples 3-12 and 16-22, add 200 μl of human plasma        (available from Bioreclamation, Inc., Hicksville, N.Y., catalog        item HMPLEDTA3) and mix the sample by pipetting.    -   5. Meanwhile, prepare samples 1, 2, 13-15, and 23 by adding, to        the other six tubes, the following: (a) for sample 1, 1 μl        luciferase control RNA and 200 μl of human plasma; (b) for        sample 13, 1 μl luciferase control RNA; (c) for sample 14, 1 μl        luciferase control RNA at a temperature of about −20° C.; (d)        for sample 23, 200 μl of human plasma; and (e) for each of        samples 2 and 15, nothing.    -   6. Allow each of samples 1-23 to sit for five minutes at about        21° C. Then, add 2 μl (1 mg) of MAGAZORB™ paramagnetic particles        to each sample and then mix each sample by pipetting.    -   7. Allow the samples to sit for three minutes at about 21° C.,        and then place the samples on a magnetic rack for two minutes.        After the two minutes, remove the supernatants from the samples,        leaving only the particles.    -   8. To each of samples 1-23, add 8 μl of blue/orange loading dye        (available from Promega, catalog item G1881) and resuspend the        particles by mixing them by pipetting.    -   9. Repeat step 8 five more times for each sample.    -   10. Using blue/orange 6× loading dye, load 8 μl of each of        samples 1-23 into a respective one of twenty-three agarose gel        electrophoresis lanes containing a 15% TBE-urea gel.    -   11. Perform electrophoresis using bromphenol blue dye at 120        volts for about 3 hours or until the bromphenol blue dye reaches        the bottom slit of the gel. Stain each lane for 15 minutes with        SYBR™ Gold, and digitally image all of the lanes using an ALPHA        INNOTECH FLUOROCHEM™ Imaging System, and, in particular, the        Amersham TYPHOON™ platform with settings of: 1. ex488/em526. 2.        PMT 450.

FIGS. 3A and 3B show the results of the electrophoresis analysis of thetwenty-three samples prepared according to Example 5. In FIG. 3A, thelanes are numbered 1-12, from left to right. In FIG. 3B, the lanes arenumbered 13-24, from left to right. Lanes 1-23 show the results forsamples 1-23, respectively. Lane 24 is empty.

As shown in FIGS. 3A and 3B, the GTC-A formulations were effective inthe purification of RNA notwithstanding the presence of certaininhibitors. Moreover, the use of certain inhibitors in addition to GTC-Aenables the selective purification of RNA, while reducing thedegradation of the RNA from RNase during the lysis process. Inparticular, GTC-A formulations containing BME alone (lanes 3-6) wereeffective in the purification of larger-sized RNA. Acting as aninhibitor of RNase, the combination of GTC-A and increasing BMEconcentrations reduced the degradation of the RNA. The GTC-Aformulations containing TCEP (lanes 7-10) also inhibited RNA degradationand resulted in less RNA being bound to the MagaZorb® paramagneticparticles than that bound using the GTC-A formulations containing BME.However, the RNA bound to the MAGAZORB™ paramagnetic particles using theGTC-A formulations containing TCEP had a higher average molecular weightthan that of the RNA bound using the GTC-A formulations containing BME.1-TG added to the GTC-A formulation (lanes 16-18 and 20) also resultedin the purification of larger-sized RNA than that obtained using theGTC-A formulations containing BME. 33% 1-TG (lane 18) demonstratedgreater inhibition of RNase than 12.5% 1-TG (lane 16). The GTC-Aformulations containing BME alone, TCEP alone, and 1-TG alone alldemonstrated effective inhibition of RNase. Similarly, the inclusion ofboth 1-TG and BME in the GTC-A formulation (lane 8) also demonstratedeffective RNase inhibition.

Luciferase control RNA added into human blood plasma (lanes 13 and 14)was bound to the MAGAZORB™ paramagnetic particles using the GTC-Aformulation. In contrast, the absence of added human plasma (lane 1)demonstrated that the luciferase RNA was not visibly degraded. However,the addition of human plasma (lane 23) resulted in RNA degradation.

Example 6

In this example, DNA as small as about 25 bp in size was purified fromhuman whole blood samples using a GTC-A formulation. The followingprocedure was used:

-   -   1. Prepare twelve samples, 1-12, by adding 200 μl of human whole        blood and 800 μl of “D₁,” the latter of which is made by adding        0.7 gm of CHAPS, 400 μl of TERGITOL™ type NP-9, and 400 μl of        TRITON™ X-100 to a 10 ml mixture of 4.3M GTC/5.9M acetamide, to        each of twelve 1.5 ml plastic tubes.    -   2. To each of samples 1-10, add 10 μl of 100 bp DNA ladder.    -   3. Mix each of samples 1-12 thoroughly by repeated pipetting.    -   4. To each of samples 1-12, add 30 μl of MAGAZORB™ paramagnetic        particles and again mix each sample by pipetting.    -   5. Incubate each sample for 20 minutes at about 21° C.    -   6. Into each of samples 1-12, insert a magnetic bar contained        within a plastic sleeve, in order to collect the MAGAZORB™        paramagnetic particles.    -   7. Remove the sleeve/magnetic bar with the collected MAGAZORB™        paramagnetic particles.    -   8. Place the sleeve/magnetic bar and the collected sample 1-12        particles into fresh tubes, each containing a 500 μl solution of        “W₁,” which is a mixture of 2.6M GTC and 7.1M acetamide.        Withdraw the magnets from the plastic sleeves, and then        resuspend the sample 1-12 particles by mixing the sleeve with        the W₁ solution. Reinsert the magnetic bar into the plastic        sleeves for a total of 30 seconds, and then remove the        sleeve/magnetic bar with the collected MAGAZORB™ paramagnetic        particles.    -   9. Repeat step 7 two more times for a total of three washes with        500 μl of W₁ for each sample.    -   10. For each of samples 1 and 2, repeat step 7 an additional        four times using W₁ for a total of seven washes with W₁.    -   11. For each of samples 3 and 4, repeat step 7 an additional        four times using 500 μl of MAGAZORB™ DNA Binding Solution        instead of W₁.    -   12. For each of samples 5 and 6, repeat step 7 an additional        four times using 500 μl of Alcohol Wash, Blood (BW wash)        (available from Promega, catalog item MB1001) solution instead        of W₁.    -   13. Place the sleeve/magnetic bar and the collected sample 1-12        particles into fresh tubes, respectively, each containing 40 μl        of WIZARD™ Genomic DNA Rehydration Solution (available from        Promega, catalog item A7963). Withdraw the magnets from the        plastic sleeves, and then resuspend the sample 1-12 particles by        mixing the sleeve with the WIZARD™ Genomic DNA Rehydration        Solution.    -   14. To each of samples 2 and 4, add 0.3 μl of 100 bp DNA (to        highlight the 100 bp band in the sample).    -   15. To each of samples 6 and 8, add 0.3 μl of 200 bp DNA (to        highlight the 200 bp band in the sample).    -   16. To each of samples 10 and 12, add 0.3 μl of 300 bp DNA (to        highlight the 300 bp band in the sample).    -   17. To each of samples 4, 5, 7, 8, 11, and 12, add 4 μl of 300        bp DNA (to highlight the 300 bp band in the sample).    -   18. Using blue/orange 6× loading dye, load 8 μl of each of        samples 1-12 into a respective one of twelve agarose gel        electrophoresis lanes containing a 15% TBE-urea gel.    -   19. Perform electrophoresis using bromphenol blue dye at 120        volts for about 3 hours or until the bromphenol blue dye reaches        the bottom slit of the gel. Stain each lane for 15 minutes with        SYBR™ Gold, and digitally image all of the lanes using an ALPHA        INNOTECH FLUOROCHEM™ Imaging System, and, in particular, the        Amersham TYPHOON™ platform with settings of: 1. ex488/em526. 2.        PMT 450.

FIG. 4 shows the results of the electrophoresis analysis of the twelvesamples prepared according to Example 6. In FIG. 4, the lanes arenumbered 1-12, from left to right. Lanes 1-12 show the results forsamples 1-12, respectively.

As shown in FIG. 4, the GTC-A formulation in conjunction with a bindingmatrix is suitable in purifying genomic DNA that is anywhere from 25bases to 20 kilobase pairs or larger. Specifically, lanes 2 and 10 showthe purification of DNA which was about 25 bases in length and showedthat oligo primers of 22 and 29 bases migrated to the bottom of the gel.Using BW wash (lanes 5 and 6) resulted in the purification oflarger-sized DNA, about 75 bp, than the 25 bp obtained without using theBW wash. Furthermore, there are DNA bands in all three purificationmethods which can be seen, for example, between the 100 bp band and 200bp band, as well as between the 200 bp and 300 bp bands, as a result ofthe added DNA bands being bound by the GTC-A formulation.

Example 7

In this example, nucleic acids from tissue culture cells were purifiedusing a GTC-A formulation. The following procedure was used:

-   -   1. Prepare six samples, 1-6, by adding 600 μl of a mixture of        4.0M GTC, 5.0M acetamide, 20% (volume/volume) 1-thio-glycerol,        0.64% (weight/volume) CHAPS, 0.64% (volume/volume) TERGITOL™        type NP-9, and 0.16% (volume/volume) TRITON™ X-100 to each of        six 1.5 ml plastic tubes.    -   2. To each of samples 1 and 2, add 200 μl of HeLa cells (2×10⁶        cells) and mix the samples by pipetting.    -   3. To sample 3, add 100 μl of HeLa cells in GIBCO™ DMEM medium        (1×10⁶ cells) (available from INVITROGEN™, catalog        item 11965092) and mix the sample by pipetting.    -   4. To each of samples 4 and 5, add 150 μl of Chinese Hamster        Ovary (CHO) cells (2×10⁶ cells) and mix the sample by pipetting.    -   5. To sample 6, add 75 μl of CHO cells in GIBCO™ F-12 plus 10%        FBS medium) (1×10⁶ cells) and mix the sample by pipetting.    -   6. Incubate each of samples 1-6 for 10 minutes at a temperature        of about 21° C. and then place the samples into a respective one        of six DNA IQ™ spin basket tubes, each containing a cellulose        membrane column.    -   7. After allowing samples 1-6 to sit for five minutes at about        21° C., centrifuge the samples for one minute at 12,000×g (times        gravity).    -   8. Remove the columns from each tube and then place the sample        1-6 columns into a respective clean 1.5 ml tube. To each of        samples 1-6, add a mixture of 500 μl of 4.0M GTC, 5.0M        acetamide, and 20% (volume/volume) 1-TG, and then centrifuge        each sample for one minute at 12,000×g.    -   9. Repeat step 8 a second time.    -   10. Repeat step 8 two more times, except substituting a 500 μl        mixture of 2.6M GTC and 7.1M acetamide for the 500 μl mixture of        4.0M GTC, 5.0M acetamide, and 20% (volume/volume) 1-TG used in        step 8.    -   11. For samples 2 and 5 only, repeat step 8 one more time,        except substituting 500 μl of SV Total RNA Wash for the 500 μl        mixture of 4.0M GTC, 5.0M acetamide, and 20% (volume/volume)        1-TG used in step 8.    -   12. Place each of samples 1-6 into respective clean 1.5 ml tubes        and add 50 μl of nuclease-free water to each tube.    -   13. Allow samples 1-6 to sit for two minutes at about 21° C.,        and then centrifuge the samples at 12,000×g for one minute.        Elute the samples in nuclease-free water in order to reduce the        amount of GTC-A carried over from the previous washes.    -   14. To maximize the nucleic acid yield, prepare six additional        samples, 7-12, by removing the columns from samples 1-6 and        placing them into respective clean 1.5 ml tubes.    -   15. Add 100 μl of nuclease-free water to each of samples 7-12,        and then incubate the samples at about 56° C. for 15 minutes.    -   16. Centrifuge samples 7-12 at 12,000×g for one minute. Elute        the samples in nuclease-free water.    -   17. Add 2 μl of 100 bp DNA ladder to sample 1 (to provide        molecular weight reference marker, thus serving as a control).    -   18. Using blue/orange 6× loading dye, load 5 μl of each of        samples 1-12 into a respective one of twelve agarose gel        electrophoresis lanes containing a 10% TBE-urea gel (available        from INVITROGEN™, Carlsbad, Calif., catalog item EC68752BOX).    -   17. Perform electrophoresis using bromphenol blue dye at 120        volts for about 3 hours or until the bromphenol blue dye reaches        the bottom slit of the gel. Stain each lane for 15 minutes with        SYBR™ Gold, and digitally image all of the lanes using an ALPHA        INNOTECH FLUOROCHEM™ Imaging System, and, in particular, the        Amersham TYPHOON™ platform with settings of: 1. ex488/em526. 2.        PMT 450.

FIG. 5 shows the results of the electrophoresis analysis of the samplesprepared according to Example 7. In FIG. 5, the lanes are numbered 1-12,from left to right. Lane 1 shows the results for sample 1; lane 2 showsthe results for sample 2; lane 3 shows the results for sample 3; lane 4shows the results for sample 7; lane 5 shows the results for sample 8;lane 6 shows the results for sample 9; lane 7 shows the results forsample 4; lane 8 shows the results for sample 5; lane 9 shows theresults for sample 6; lane 10 shows the results for sample 10; lane 11shows the results for sample 11; and lane 12 shows the results forsample 12.

As shown in FIG. 5, in the case of tissue culture cells, the use of theGTC-A formulation allowed the purification of genomic DNA and total RNAfrom each of the samples prepared according to Example 7.

Example 8

In this example, cytoplasmic RNA from tissue culture cell samples waspurified using GTC-A formulations. The following procedure was used:

-   -   1. Grow Human Embryonic Kidney 293 (HEK293) tissue culture cells        in GIBCO™ DMEM medium. Allow 4 ml of 5×10⁶ HEK293 cells to        settle at 1×g, and then remove the growth medium by pipetting,        so that about 2×10⁷ cells are contained in 50 μl. Next, place        the 50 μl of cells into a 1.5 ml plastic tube and place on ice        for 5 minutes.    -   2. Add 6 μl of RNASIN™ Plus (40 units per μl) (available from        Promega, catalog item 1232) to the 50 μl of cells from step 1.        Then add 100 μl of 120 μg/ml digitonin in 100 mM EDTA 100 mM        HEPES™ (pH 7.5) (on ice) to the cells. Mix the contents by        vortexing for 10 seconds, and then incubate on ice for 20        minutes. The incubation with 77 μg/ml digitonin promotes the        lysis of the cytoplasmic membrane, without lysis of the cell        nuclear membrane.    -   3. Centrifuge the suspension from step 2 at 13,000×g for five        minutes at about 4° C. to pellet the nuclei and cellular debris.    -   4. Prepare four samples, 1-4, by dispensing 40 μl of the        supernatant of the suspension from step 3 into 1.5 ml plastic        tubes containing the following: (a) for each of samples 1 and 2,        400 μl of a mixture of 4.0M GTC, 5.0M acetamide, 20%        (volume/volume) 1-TG, and 10 μl of MAGAZORB™ paramagnetic        particles; and (b) for each of samples 3 and 4, a DNA-IQ™ column        and 400 μl of a mixture of 4.0M GTC, 5.0M acetamide, and 20%        (volume/volume) 1-TG.    -   5. Mix each of samples 3 and 4 by pipetting, allow them to        settle for 20 seconds at a temperature of about 21° C., and then        spin samples 3 and 4 at 13,000×g for one minute. Remove the        column flowthrough of samples 3 and 4 and place them into fresh        1.5 ml tubes. Then, add 10 μl of MAGNESIL™ Blue to the column        flowthrough of samples 3 and 4.    -   6. Mix the four samples from step 5 by pipetting and incubating        them at about 21° C. for five minutes. Place each of samples 1-4        on a magnetic. After separation of the particles from the        samples, discard the supernatants from the particles that are        attracted to the side of the tubes.    -   7. Wash the remaining particles of each of samples 1-4 with 400        μl of a mixture of 4.0M GTC, 5.0M acetamide, and 20%        (volume/volume) 1-TG.    -   8. Place the samples containing magnetic particles on magnetic        stands, magnetize, and discard the supernatants from the        particles that are attracted to the side of the tubes.    -   9. Transfer the DNA-IQ™ columns from step 5 to clean 1.5 ml        tubes. Wash the columns using 400 μl of SV RNA Wash Solution.        Repeat this wash process an additional time for samples 2-4.    -   10. Place all four samples containing paramagnetic particles on        a magnetic stand, discard the supernatants, and allow the        samples to air dry for 10 minutes.    -   11. Place the DNA-IQ™ columns into clean 1.5 ml tubes and number        these as samples 5 and 6.    -   12. Elute each of samples 1-6 with 40 μl of nuclease-free water        per sample for 10 minutes at a temperature of about 21° C. Place        the samples containing paramagnetic particles on a magnetic        rack.    -   13. Using blue/orange 6× loading dye, load 10 μl of sample from        each tube into separate agarose gel electrophoresis lanes        containing a 15% TBE-urea gel.    -   14. Perform electrophoresis using bromphenol blue dye at 120        volts for about 3 hours or until the bromphenol blue dye reaches        the bottom slit of the gel. Stain each lane for 15 minutes with        SYBR™ Gold, and digitally image all of the lanes using an ALPHA        INNOTECH FLUOROCHEM™ Imaging System, and, in particular, the        Amersham TYPHOON™ platform with settings of: 1. ex488/em526. 2.        PMT 450.

FIG. 6 shows the results of the electrophoresis analysis of the samplesprepared according to Example 8. Lane 1 shows a 100 bp DNA ladder forthe purpose of comparing a DNA sequence; lane 2 shows the results of 10μl of initial cytoplasmic RNA supernatant to serve as a marker of nonpurified nucleic acid; lane 3 shows the results for sample 3; lane 4shows the results for sample 4; lane 5 shows the results for sample 1;lane 6 shows the results for sample 2; lane 7 shows the results forsample 5; lane 8 shows the results for sample 6; lane 9 shows 100 bp DNAladder for the purpose of comparing a DNA sequence; lane 10 shows theresults for sample 7; lane 11 shows the results for sample 8; and lane12 is empty.

As demonstrated in FIG. 6, RNA is purified using the GTC-acetamideformulation and SV RNA Wash, using either MAGAZORB™ paramagneticcellulose particles or MAGNESIL™ paramagnetic silica particles. Inaddition, ribosomal RNA and tRNA bands are visible in lanes 7 and 8,showing purification using GTC-A. Furthermore, the passage of thecytoplasmic RNA sample through a cellulose column (lanes 3 and 4), witha short sample incubation time of 20 seconds at a temperature of about21° C., substantially removed nuclear DNA. This was accomplished withoutsubstantial removal of the cytoplasmic RNA, demonstrated by thesubsequent purification using MAGNESIL™ particles (lanes 10 and 11).

Example 9

In this example, DNA or RNA from tissue culture cell samples waspurified using a GTC-A formulation with the aid of various bindingmatrices in order to determine additional binding matrices suitable foruse with the present invention. The following procedure was used:

-   -   1. Centrifuge 2.3×10⁷ HEK293 cells, grown in DMEM medium, at        1000×g in a 50 ml plastic tube for five minutes. Decant the        supernatant and mix the pelleted cells by vortexing.    -   2. In order to lyse the HEK293 cells, add 8 ml of a mixture of        4.0M GTC, 5.0M acetamide, 20% (volume/volume) 1-TG, 0.64%        (weight/volume) CHAPS, 0.64% (volume/volume) TERGITOL™ type        NP-9, and 0.16% (volume/volume) TRITON™ X-100. Mix the lysate by        vortexing until a uniform mixture is obtained.    -   3. Prepare twenty-four samples, 1-24, by adding the indicated        one of the following binding matrices to a 1.5 ml plastic        tube: (a) for samples 1 and 2, a cellulose membrane in a DNA-IQ™        column; (b) for samples 3 and 4, a silica membrane in a SV        column; (c) for samples 5 and 6, a cellulose acetate membrane in        a CORNING™ spin column; (d) for samples 7 and 8, a nylon        membrane in a CORNING™ spin column; (e) for samples 9 and 10, a        PVDF membrane in a polypropylene spin column; (f) for samples 11        and 12, a polypropylene membrane in a DNA-IQ™ column; (g) for        samples 13 and 14, a HIGH PURE™ Spin Filter tube; (h) for        samples 15 and 16, a clearing column; (i) for samples 17 and 18,        10 μl (3.4 mg) of paramagnetic zeolite particles; (j) for        samples 19 and 20, 20 μl (1 mg) of MAGAZORB™ paramagnetic        particles; (k) for samples 21 and 22, 10 μl (1 mg) of MAGNESIL™        Blue; and (1) for samples 23 and 24, 50 μl (5 mg) of DNA-IQ™        paramagnetic particles.    -   4. Add 300 μl of HEK293 lysate to each of samples 1-24.    -   5. Incubate all of samples 1-24 at about 21° C. for 10 minutes.        Then place samples 17-24, which contain particles, on a magnetic        rack in order to separate the particles from the supernatants.        Then discard the supernatants.    -   6. Centrifuge column samples 1-16 at 13,000×g for five minutes.        Note that the PVDF columns (samples 9 and 10) allow only about        half of the solution to flow through the membrane. Thus, the        unprocessed fluid is to be discarded along with the        flowthroughs.    -   7. Resuspend each of particle samples 17-24 in 400 μl of a        mixture of 1.7M GTC and 7.5M acetamide by pipetting, and then        place the particle samples back on the magnetic rack. Discard        the supernatants.    -   8. Repeat step 7 three more times using 400 μl of SV RNA Wash        Solution instead of the GTC-A formulation. After discarding the        third RNA wash solution, air-dry the particle samples for 20        minutes at a temperature of about 21° C. Then elute the nucleic        acids in 40 μl of nuclease-free water.    -   9. Wash each of column samples 1-16 with a 400 μl mixture of        1.7M GTC and 7.5M acetamide, followed by centrifugation at        13,000×g for five minutes. Discard the column flowthroughs.    -   10. Wash each of column samples 1-16 twice with 400 μl of SV RNA        Wash Solution, followed by centrifugation at 13,000×g for five        minutes, and then discard the column flowthroughs. Then, spin        the column samples at 13,000×g for one minute to ensure the        columns are dry, and then elute each column sample with 40 μl of        nuclease-free water.    -   11. Store all of samples 1-24 at about −20° C. for 16 hours.    -   13. After magnetic separation of samples 17-24, load 10 μl of        each sample into a respective one of twenty-four agarose gel        electrophoresis lanes containing a 15% TBE-urea gel, using        blue/orange 6× loading dye.    -   14. Perform electrophoresis using bromphenol blue dye at 120        volts for about 3 hours or until the bromphenol blue dye reaches        the bottom slit of the gel. Stain each lane for 15 minutes with        SYBR™ Gold, and digitally image all of the lanes using an ALPHA        INNOTECH FLUOROCHEM™ Imaging System, and, in particular, the        Amersham TYPHOON™ platform with settings of: 1. ex488/em526. 2.        PMT 450.

FIGS. 7A and 7B show the results of the electrophoresis analysis of thesamples prepared according to Example 9. In FIG. 7A, the lanes arenumbered 1-12, from left to right. In FIG. 7B, the lanes are numbered13-24, from left to right. Lanes 1-24 show the results for samples 1-24,respectively.

As shown in FIGS. 7A and 7B, genomic DNA was purified using celluloseacetate columns, PVDF columns, DNA-IQ™ paramagnetic particles, andMAGNESIL™ Blue particles. In addition, MAGAZORB™ paramagnetic particlespurified a small amount of RNA as well as genomic DNA. Both RNA and DNAwere purified using SV silica columns, nylon membrane columns,polypropylene membrane columns, HIGH PURE™ Spin columns, clearingcolumns, and paramagnetic zeolite particles, which showed prominent tRNAbands.

Furthermore, this example shows how to screen for the initial binding ofnucleic acids to a binding matrix, followed by the retention of nucleicacids during sequential washes, and elution of the nucleic acids at theend of the procedure. For example previous examples have shown bindingof both DNA and RNA to MAGAZORB™ paramagnetic particles, and in thisexample (with washes using SV RNA Wash) the RNA was less prominent inthe elution. Therefore, in order to screen binding matrices for bindingof nucleic acids, one could wash only with the initial GTC-A formulationto ensure retention of nucleic acids on the binding matrix. This exampledemonstrated a measure of the ability to retain the nucleic acids on thebinding matrix during wash steps. Additionally, this example alsorequired that the nucleic acid was eluted from the binding matrix. Inaddition to the requirement that the binding matrix has bound DNA orRNA, the nucleic acid must also remain bound during washes with twodifferent solutions, and also allows the nucleic acid to elute from thebinding matrix at the end of the purification. Even with all threerequirements, it is shown that all of the binding matrices show anability to purify nucleic acid(s) using GTC-A formulations.

Example 10

In this example, DNA and RNA from the same tissue culture cell sampleswere purified using a GTC-A formulation. The following procedure wasused:

-   -   1. Centrifuge 1.6×10⁷HEK293 cells grown in DMEM medium at 1000×g        in a 15 ml plastic tube for five minutes.    -   2. Lyse the cells adding 6 ml of D_(X). Then mix the lysate by        repeated pipetting until a uniform mixture is obtained.    -   3. Prepare two samples, 1 and 2, by adding 400 μl of the lysate        from step 2 to each of two 1.5 ml plastic tubes, each containing        50 μl (5 mg) of DNA-IQ™ particles. Mix the samples, and then        incubate for 10 minutes at about 21° C.    -   4. Magnetize the particles in each of samples 1 and 2 using a        magnetic rack.    -   5. Prepare two additional samples, 3 and 4, by transferring the        supernatants from samples 1 and 2, respectively, to clean tubes,        each containing 10 μl (3.4 mg) of paramagnetic zeolite        particles. Samples 1 and 2 now consist of the DNA-IQ™ particles        minus the supernatants.    -   6. Mix and incubate each of samples 3 and 4 for 10 minutes at a        temperature of about 21° C. Next, magnetize the particles in        each of samples 3 and 4 using a magnetic rack and remove the        supernatants from each of samples 3 and 4.    -   7. Wash each of samples 1-4 with 800 μl of a mixture of 4.0M        GTC, 5.0M acetamide, and 20% (volume/volume) 1-TG. Then wash        each of the samples an additional three times with 800 μl of SV        RNA Wash Solution.    -   8. Magnetize the particles in samples 1-4, remove the        supernatants, and then air dry the particles for 20 minutes at a        temperature of about 21° C.    -   9. Elute the particles in each of samples 1-4 in 40 μl of        nuclease-free water for ten minutes at a temperature of about        21° C. in order to allow the nucleic acids to elute from the        particles.    -   10. Using blue/orange 6× loading dye, load 10 μl of each of        samples 1-4 into a respective one of four agarose gel        electrophoresis lanes containing a 15% TBE-urea gel.    -   11. Perform electrophoresis using bromphenol blue dye at 120        volts for about 3 hours or until the bromphenol blue dye reaches        the bottom slit of the gel. Stain each lane for 15 minutes with        SYBR™ Gold, and digitally image all of the lanes using an ALPHA        INNOTECH FLUOROCHEM™ Imaging System, and, in particular, the        Amersham TYPHOON™ platform with settings of: 1. ex488/em526. 2.        PMT 450.

FIG. 8 shows the results of an agarose gel electrophoresis analysis ofthe samples prepared according to Example 10. In FIG. 8, the lanes arenumbered 1-12, from left to right. Lanes 1 and 2 show the results forsamples 1 and 2, respectively; lane 3 is blank; lanes 4 and 5 showsamples 1 and 2, respectively, digested with RNase ONE; lane 6 containsPromega 100 bp DNA ladder; lanes 7 and 8 show results for samples 3 and4, respectively; lane 9 is blank; lanes 10 and 11 show samples 3 and 4,respectively, digested with Promega RQ1 RNase-free DNase; and lane 12contains Promega genomic DNA.

As shown in FIG. 8, genomic DNA was purified using DNA-IQ™ particles,and from the same medium, RNA was purified using paramagnetic zeoliteparticles. Thus, DNA and RNA can be purified from the same medium usinga first binding matrix to bind DNA and a second binding matrix to bindRNA, without adding any additional solution or condition between thefirst binding matrix and the application of the second binding matrix.

Example 11

In this example, RNA from tissue culture cell mediums were purifiedusing GTC-A formulations containing GTC and acetamide, N-methylacetamideor N,N-dimethylacetamide. The following procedure was used:

-   -   1. Allow 25 ml of 1.5×10⁶ HEK293 tissue culture cells, grown in        GIBCO™ DMEM medium, to settle at 1×g for sixty minutes at about        21° C., and then remove the growth medium supernatant by        pipetting, so that about 3.7×10⁷ cells are contained in 1.5 ml        (about 2.5×10⁶ cells per 100 μl).    -   2. Prepare twelve samples, 1-12, by adding the following to a        respective one of twelve 1.5 ml plastic tubes: (a) for samples        1-6, 100 μl of HEK293 tissue culture cells; and (b) for samples        7-12, 100 μl of human whole blood.    -   3. To each of samples 2, 4, 6, 8, 10, and 12, add 10 μl of        Promega 100 base pair ladder.    -   4. To each sample, add the indicated GTC-A formulation: (a) for        each of samples 1, 2, 7, and 8, 300 μl of a mixture of 4.0M GTC,        5.0M acetamide, and 20% (volume/volume) 1-TG; (b) for each of        samples 3, 4, 9, and 10, 500 μl of a mixture of 4.0M GTC, 5.0M        N-methylacetamide, and 10% (volume/volume) 1-TG; and (c) for        each of samples 5, 6, 11, and 12, 500 μl of a mixture of 4.0M        GTC, 5.0M N,N-dimethylacetamide, and 10% (volume/volume) 1-TG.    -   5. Mix each of samples 1-12 by vortexing, and then incubate the        samples for five minutes at about 21° C.    -   6. Transfer each of samples 1-12 to one a respective 1.5 ml        plastic tube containing DNA-IQ™ cellulose mini-columns, and then        centrifuge the samples at 11,000×g for two minutes.    -   7. Transfer each of samples1-12 DNA-IQ™ cellulose mini-columns        to fresh 1.5 ml tubes, then wash each mini-column with 500 μl of        the same GTC-A formulation used in step 4.    -   8. Elute each of samples 1-12 with 40 μl of nuclease-free water        and then remove the mini-columns.    -   9. Prepare twelve additional samples, 13-24, by placing the        mini-columns removed from samples 1-12, respectively, into fresh        1.5 ml tubes.    -   10. Elute each of samples 13-24 with 40 μl of nuclease-free        water and then discard the mini-columns.    -   11. Using blue/orange 6× loading dye, load 10 μl of each of        samples 1-24 into a respective one of twenty-four agarose gel        electrophoresis lanes containing a 15% TBE-urea gel.

12. Perform electrophoresis using bromphenol blue dye at 120 volts forabout 3 hours or until the bromphenol blue dye reaches the bottom slitof the gel. Stain each lane for 15 minutes with SYBR™ Gold, anddigitally image all of the lanes using an ALPHA INNOTECH FLUOROCHEM™Imaging System, and, in particular, the Amersham TYPHOON™ platform withsettings of: 1. ex488/em526. 2. PMT 450.

FIGS. 9A and 9B show the results of the electrophoresis analysis of thesamples prepared according to Example 11. In FIG. 9A, the lanes arenumbered 1-12, from left to right. In FIG. 9B, the lanes are numbered13-24, from left to right. Lanes 1-24 show the results for samples 1-24,respectively.

As shown in FIGS. 9A and 9B, DNA and RNA are bound to cellulosemembranes in mini-columns, and purified using GTC and acetamide, GTC andN-methylacetamide, and GTC and N,N-dimethylacetamide, showing thatderivatives of acetamide can also be used in conjunction with GTC topurify nucleic acids.

Example 12

In this example, DNA purified from human whole blood was bound to ablood card, which served as a binding matrix. Additionally, the bloodsample can be washed on the blood card, and purified nucleic acids canbe obtained by elution of blood card punches. The following procedurewas used:

-   -   1. Prepare two samples, 1 and 2, by adding 200 μl of a mixture        of 4.0M GTC, 5.0M acetamide, 20% (volume/volume) 1-TG, 0.64%        (weight/volume) CHAPS, 0.64% (volume/volume) TERGITOL™ type        NP-9, and 0.16% (volume/volume) TRITON™ X-100 to each of two 1.5        ml plastic tubes.    -   2. Add 100 μl of human whole blood to each of samples 1 and 2.    -   3. To sample 2, add 10 μl of Promega 100 bp ladder.    -   4. Mix each of samples 1 and 2 thoroughly by pipetting at a        temperature of about 21° C.    -   5. In 10 μl increments, spot each of samples 1 and 2 onto a        Schleicher & Schuell cellulose card, with successive additions        being applied only after the previous addition has been absorbed        by the cellulose card. Continue until the entire sample (300 μl        for sample 1 and 310 μl for sample 2) has been applied to the        cellulose card.    -   6. Spot 100 μl of human whole blood plus 10 μl of RNA Marker        (available from Promega, catalog item G3191) onto the cellulose        card in order to compare the nucleic acids.    -   7. To the sample 2 spot, apply 150 μl of a mixture of 2.6M GTC        and 7.1M acetamide in 10 μl increments, with successive        additions being applied only after the previous addition has        been absorbed by the cellulose card. The resulting blood spot        should have a relatively white center, and a concentric red        circle containing hemoglobin and other cellular debris.    -   8. Take a central punch of about 2 mm×2 mm from the center of        each of the sample 1 and 2 spots, and then take a second punch        of about 2 mm×2 mm from about 5 mm outside of each center.    -   9. Elute each the four punches in 20 μl of nuclease-free water.    -   10. Using blue/orange 6× loading dye, load 10 μl of the elution        from each of the four punches into a respective one of four        agarose gel electrophoresis lanes containing a 15% TBE-urea gel.    -   11. Perform electrophoresis using bromphenol blue dye at 120        volts for about 3 hours or until the bromphenol blue dye reaches        the bottom slit of the gel. Stain each lane for 15 minutes with        SYBR™ Gold, and digitally image all of the lanes using an ALPHA        INNOTECH FLUOROCHEM™ Imaging System, and, in particular, the        Amersham TYPHOON™ platform with settings of: 1. ex488/em526. 2.        PMT 450.

FIG. 10 shows the Schleicher & Schuell cellulose card prepared accordingto Example 12. The spot on the left corresponds to sample 1; the spot inthe middle corresponds to sample 2; and the spot on the rightcorresponds to the blood plus RNA Marker.

FIG. 11 shows the results of the electrophoresis analysis of the fourelutions prepared according to Example 12. In FIG. 11, the lanes arenumbered 1-12, from left to right. Lane 1 shows the results for thecenter punch of sample 1; lane 3 shows the results for the second punchof sample 1; lane 5 shows the results for the center punch of sample 2;lane 7 shows the results for the second punch of sample 2; lane 9 showsPromega 100 bp DNA Ladder; lane 11 shows Promega genomic DNA standard;and the remaining lanes are empty.

As shown in FIG. 11, substantially more DNA was obtained from the centerpunches than from the second punches for each of samples 1 and 2.Furthermore, DNA of sizes down to 50 base pairs can be retained in thecentral blood application spot and be eluted with nuclease-free water.

Although this invention has been described in certain specific exemplaryembodiments, many additional modifications and variations would beapparent to those skilled in the art in light of this disclosure. It is,therefore, to be understood that this invention may be practicedotherwise than as specifically described. Thus, the exemplaryembodiments of the invention should be considered in all respects to beillustrative and not restrictive, and the scope of the invention to bedetermined by any claims supported by this application, and theequivalents thereof, rather than by the foregoing description.

What is claimed:
 1. A method of purifying a nucleic acid present in an in vivo cellular environment, the nucleic acid and in vivo cellular environment being contained in a medium, the method comprising: (a) combining a medium containing a nucleic acid with a binding matrix and an aqueous formulation, the formulation comprising: i) guanidine thiocyanate or guanidine hydrochloride; and ii) acetamide, or one or more acetamide derivatives, or a combination of acetamide and one or more acetamide derivatives, the amounts of the guanidine thiocyanate or guanidine hydrochloride and the acetamide, the acetamide derivative(s), or the combination of acetamide and acetamide derivative(s) present in the formulation being sufficient to cause the nucleic acid to separate from its in vivo cellular environment and bind to the binding matrix; (b) separating the binding matrix with the nucleic acid bound thereto from substantially the rest of the combined medium and formulation; and (c) eluting the nucleic acid from the binding matrix, thereby obtaining the nucleic acid in a substantially purified form.
 2. The method of claim 1, wherein the acetamide derivative(s) are selected from the group consisting of N-methylacetamide and N,N-dimethylacetamide.
 3. The method of claim 1, wherein the concentration of the guanidine thiocyanate or guanidine hydrochloride in the formulation is from approximately 1.7M to approximately 4.3M, and the concentration of the acetamide, the acetamide derivative(s), or the combination of acetamide and acetamide derivative(s) in the formulation is from approximately 5.0M to approximately 7.5M.
 4. The method of claim 1, wherein a second nucleic acid is present in the medium and a second binding matrix is provided in step (a), wherein the binding matrix is capable of binding the nucleic acid and the second binding matrix is capable of binding the second nucleic acid.
 5. The method of claim 1, wherein the binding matrix comprises one or more materials selected from the group consisting of paramagnetic cellulose particles, paramagnetic carboxy-cellulose particles, paramagnetic citrus pectin particles, paramagnetic apple pectin particles, paramagnetic zeolite particles, paramagnetic silica particles, cellulose membranes, silica membranes, cellulose acetate columns, nylon membrane columns, PVDF membrane columns, polypropylene columns, pure spin columns, and clearing columns.
 6. The method of claim 1, wherein step (a) further comprises combining one or more additional ingredients with the medium, the binding matrix, and the formulation, the one or more additional ingredients being selected from the group consisting of proteinase K, beta-mercaptoethanol, tris(carboxyethyl)phosphine, dithiothreitol, 1-thioglycerol, digitonin, lysis solutions, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 26-(4-nonylphenoxy)-3,6,9,12,15,18,21,24-octaoxahexacosan-1-ol, and 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol.
 7. A method of binding a nucleic acid present in an in vivo cellular environment, the nucleic acid and in vivo cellular environment being contained in a medium, to a binding matrix, the method comprising combining a medium containing the nucleic acid with the binding matrix and an aqueous formulation, the formulation comprising: (a) guanidine thiocyanate or guanidine hydrochloride; and (b) acetamide, or one or more acetamide derivatives, or a combination of acetamide and one or more acetamide derivative(s); the amounts of the guanidine thiocyanate or guanidine hydrochloride, the acetamide, the acetamide derivative(s), or the combination of acetamide and acetamide derivative(s) present in the formulation being sufficient to cause the nucleic acid to separate from its in vivo cellular environment and bind to the binding matrix.
 8. The method of claim 7, wherein the acetamide derivative(s) are selected from the group consisting of N-methylacetamide and N,N-dimethylacetamide.
 9. The method of claim 7, wherein the concentration of the guanidine thiocyanate or guanidine hydrochloride in the formulation is from approximately 1.7M to approximately 4.3M, and the concentration of the acetamide, the acetamide derivative(s), or the combination of acetamide and acetamide derivative(s) in the formulation is from approximately 5.0M to approximately 7.5M.
 10. The method of claim 9, wherein the concentration of the guanidine thiocyanate or guanidine hydrochloride in the formulation is from approximately 4.0M to approximately 4.3M, and the concentration of the acetamide, the acetamide derivative(s), or the combination of acetamide and acetamide derivative(s) in the formulation is from approximately 5.0M to approximately 7.1M.
 11. The method of claim 7, wherein the ratio of the formulation to the medium is from 1:1 to 30:1, by volume, and the ratio of the binding matrix to the medium is from 0.005:1 to 0.5:1, by volume.
 12. The method of claim 11, wherein the ratio of the formulation to the medium is from 1.5:1 to 8:1, by volume, and the ratio of the binding matrix to the medium is from 0.2:1 to 0.4:1, by volume.
 13. The method of claim 7, wherein the binding matrix comprises one or more materials selected from the group consisting of paramagnetic cellulose particles, paramagnetic carboxy-cellulose particles, paramagnetic citrus pectin particles, paramagnetic apple pectin particles, paramagnetic zeolite particles, paramagnetic silica particles, cellulose membranes, silica membranes, cellulose acetate columns, nylon membrane columns, PVDF membrane columns, and polypropylene columns.
 14. A kit comprising a binding matrix and an aqueous formulation, the formulation comprising an amount of guanidine thiocyanate or guanidine hydrochloride and an amount of (i) acetamide, or (ii) one or more acetamide derivatives, or (iii) a combination of acetamide and one or more acetamide derivatives, the amounts of the guanidine thiocyanate and the acetamide, the acetamide derivative(s), or the combination of acetamide and acetamide derivative(s) present in the formulation being sufficient to cause a nucleic acid present in an in vivo cellular environment, the nucleic acid and in vivo cellular environment contained in a medium, to separate from its in vivo cellular environment and to bind to the binding matrix, when the medium containing the at least one nucleic acid is combined with the binding matrix and the formulation.
 15. The kit of claim 14, wherein the concentration of the guanidine thiocyanate or guanidine hydrochloride in the formulation is from approximately 1.7M to approximately 4.3M, and the concentration of the acetamide, acetamide derivative(s), or the combination of acetamide and acetamide derivative(s) in the formulation is from approximately 5.0M to approximately 7.5M.
 16. The kit of claim 15, wherein the concentration of the guanidine thiocyanate in the formulation is from approximately 4.0M to approximately 4.3M, and the concentration of the acetamide, the acetamide derivative(s), or the combination of acetamide and acetamide derivative(s) in the formulation is from approximately 5.0M to approximately 7.1M.
 17. The kit of claim 14, wherein the ratio of the binding matrix to the formulation in the kit is from 1:400 to 1:1, by volume. 